Abstract

From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.

Cdt1 interacts with SNF2H.A, nuclear extracts were prepared from HEK293T cells and immunoprecipitated with anti-Cdt1 antibody (Cdt1 IP) or control rabbit IgG (cont IP). Immunoprecipitates were subjected to immunoblotting with the indicated antibodies. Three percent of the input was also loaded. B, HEK293T cells were transfected with T7-Cdt1 and SNF2H expression vectors, and nuclear extracts were prepared. After immunoprecipitation with anti-SNF2H antibody, the precipitates were blotted with the indicated antibodies. One percent of the input for SNF2H or 0.3% of the input for Cdt1 was loaded. C, HEK293T cells were transfected with T7-Cdt1 and SNF2H expression vectors, and nuclear extracts were prepared. After immunoprecipitation with anti-Cdt1 antibody, the precipitates were immunoblotted with the indicated antibodies. One percent of the input was loaded. D, HEK293T cells were transfected with the indicated expression vectors, and nuclear extracts were prepared. After immunoprecipitation with anti-Cdt1 antibody or control rabbit IgG, the precipitates were immunoblotted with the indicated antibodies. Three percent of the input was loaded. E, GST-Cdt1 or GST was incubated with SNF2H protein synthesized by in vitro transcription-translation with rabbit reticulocyte lysate. GST-Cdt1 and the associated SNF2H protein were then collected on glutathione beads and subjected either to immunoblotting with anti-SNF2H antibody (upper panel) or to Coomassie Brilliant Blue staining (lower panel). Twenty percent of the input sample was analyzed. F, GST-Cdt1 or GST was incubated with HeLa nuclear extracts, and bound proteins were analyzed by immunoblotting with the indicated antibodies. Fifteen percent of the input was loaded.

Silencing of SNF2H suppresses MCM, but not Cdt1, loading at origins.A–C, HeLa cells transfected with control (GL2; siCont) or SNF2H (siSNF2H) siRNAs for 48 h were subjected to immunoblotting (A), ChIP assay (B), or FACS analysis (C). Coomassie Brilliant Blue (CBB) staining served as the loading control. For ChIP data, the means ± S.D. are shown (n = 3). *, p < 0.05. D–F, T98G cells were transfected with the siRNAs for 24 h, rendered quiescent by serum starvation for 48 h, and then stimulated to reenter the cell cycle by serum addition. Cells were harvested at the indicated times and subjected to chromatin binding assay (D and E) or FACS analysis (F). D, whole cell extracts and chromatin/matrix-binding fractions were immunoblotted with the indicated antibodies. E, the signal intensities of the pre-RC proteins were quantified, normalized to the signals of Coomassie Brilliant Blue staining, and are shown with the maximal values set at 100%. The means ± S.D. are shown (n = 2).

Cdt1 overexpression-induced ATM activation is decreased by SNF2H silencing. HEK293T cells were transfected with the T7-Cdt1 Cy+D1m expression vector and SNF2H siRNAs using Lipofectamine 2000 along with a plasmid expressing GFP to allow monitoring of the transfection efficiency. GL2 siRNAs were used as the control. Whole cell lysates were prepared 48 h after transfection and immunoblotted with the indicated antibodies. The signal intensity of each band was quantified, with the intensity of the band corresponding to cells transfected with T7-Cdt1 and treated with control siRNAs set at 100%. The means ± S.D. are shown (n = 3).